The increasing incidence of multi-drug resistance in Staphylococcus aureus and other bacteria represents a public health crisis. Two thirds of hospital-associated S. aureus infections and ~50% of those acquired in the community are now methicillin-resistant (MRSA). MRSA causes >450,000 infections in the US each year, and it is responsible for half of all US deaths caused by drug-resistant bacteria. This threat to public health is creating demand for new therapeutic agents, but traditional antibiotic development pipelines are not keeping pace with the escalating problem. Moreover, antibacterial chemotherapies have proven widely susceptible to rapid evolution of bacterial resistance. Bacteriolytic enzymes, such as Staphylococcus simulans lysostaphin, are an innovative new class of antibiotics that catalytically dismantle cell wall peptidoglycan causing bacterial lysis and death. Due to peptidoglycan?s conserved nature and complex biosynthesis, such enzymes have proven less susceptible to emergent resistance. Unfortunately, lysostaphin elicits anti-drug antibodies in vivo, and this immunogenicity and associated toxicity are barriers to clinical translation. Supported by a successful Phase I STTR grant, Stealth Biologics has re-engineered lysostaphin for reduced immunogenicity in humans. The pivotal outcomes of the Phase I STTR project were: 1) design and construction of F12, a globally deimmunized variant of lysostaphin; 2) in vitro validation of F12?s anti-MRSA potency; 3) preliminary quantification of F12 synergy with FDA approved antibiotics; 4) demonstration of reduced immunogenicity in human cellular immunoassays; and 5) confirmation of reduced in vivo immunogenicity and consequent enhanced therapeutic efficacy in humanized HLA transgenic mice. Collectively, these data suggest that F12 is a promising therapeutic for drug-resistant S. aureus infections. In the proposed Phase II STTR, we have designed a systematic and focused strategy for constructing an F12 target product profile (TPP). In Aim 1, F12 manufacturing and purification will be optimized and scaled up. In Aim 2, an initial clinical indication will be selected based on F12?s in vivo efficacy in two well-established models: rabbit bacteremia/endocarditis and murine skin infection. Efficacy studies will be supported by rigorous experimental analyses of in vitro potency, in vitro resistance susceptibility, in vivo maximum tolerated dose, and in vivo pharmacokinetics. Aim 3 will yield a preliminary toxicity and immunogenicity profile in rabbits and humanized mice that have received escalating single or repeated doses of F12. The resulting data package will enable construction of a TPP that will guide design and execution of comprehensive IND-enabling studies. We anticipate that F12-based antibacterial therapies will prove to be potent, safe, and amenable to repeated dosing. As such, F12 will benefit from competitive advantages relative to more immunogenic phage endolysins, and ultimately it may represent a breakthrough drug for life-threatening MRSA infections.